Current technologies for polynucleic acid analysis are diverse and include PCR (polymerase chain reaction) and quantitative PCR, RNA- or cDNA-microarrays, 3′- and 5′ RACE, rapid amplification of cDNA ends, cap analysis of gene expression, CAGE, serial analysis of gene expression, SAGE, cloning experiments, Sanger sequencing, massively parallel sequencing also known as next generation sequencing, NGS and SQUARE, a primer matrix based segregation method which groups the transcriptome into classes of molecules which share the same start- and end sequences [Seitz, 2007]. The field of protein analysis methods is much wider. Complex proteoms are studied by 2D and 3D electrophoresis, which separate proteins due to their physical properties, mass spectrometry for the high-throughput identification of proteins and sequencing of peptides, protein microarrays to detect relative protein concentrations, or two-hybrid screening for the exploration of protein-protein and nucleic acid-protein interactions.
NGS technologies are tag-based methods to sequence genomes and transcriptomes. One example is its combination with RNA-Seq which extracts random short sequence fragments from RNA molecules. Despite obvious advantages like hypothesis neutrality, which means that this method can be applied without pre-knowledge, several drawbacks are evident. First, NGS systems and kits are costly in terms of manpower, time, material consumption and data processing power. Second, detection of low gene expression levels is only possible if samples are sequenced to extreme depth, which means in turn that common transcripts are read manifold before rare reads become visible. Third, the detection of transcript isoforms remains probabilistic and heavily relies on a-priori information. Independent of the applied read depth many reads cannot be uniquely assigned to the correct transcript as soon as alternatives are possible. The transcript pattern remains diffuse and often gene expression patterns, which group all gene related transcripts, are presented instead of transcriptome patterns. It contributes to the reduced applicability of NGS methods in the analysis of highly complex samples.
One hurdle lies in the entropy of systems as a measure of disorder or randomness of its constituents. Characterizing disordered systems requires identifying all individual constituents and assigning them to one group of equals, e.g. to the correct transcript. Methods which segregate the constituents in complex systems reduce their entropy. This implies that after segregation information can be gained easier because then it only requires identifying the group and the number of constituents per group. Such methods are described below on the example of transcriptome analysis
For microarray experiments different oligonucleotide probes are immobilized in separate spots on a supporting solid surface. By these means, microarrays allow to separate samples into different classes of molecules due to the hybridization of analytes to those probes. One major drawback is their lack of discriminatory power to distinguish cognate sequences, e.g. splice variants. Molecules which carry the same sequence anywhere in the molecule equally hybridize to the same probes. Cross-hybridizations are common and blur the results.
The use of two probes, and in particular two probes which have been designed as primers for PCR amplification reactions, helps to alleviate the problems of such cross-hybridization. PCR measures the presence of individual sequences. Measurements of a moderate number of different analytes in a mixture can be achieved in parallel fluid phase formats for example using multi-well PCR plates. However, the number of targeted analytes is much too small to investigate complex transcriptomes in depth.
Bridge PCR combines the multiplexing capabilities of microarrays with the accuracy and sensitivity of PCR-based assays. Here, the immobilization of primer pairs enables solid phase supported reactions. The two primers are immobilized to a single sensor surface either as a mixture or sequentially to form a mixed layer of primers at the surface which becomes very challenging for larger arrays. If a target molecule binds to such a surface it can initiate a seed for amplification and in succession a surface mediated PCR reaction. One difficulty results from the fact that different sites of the analyte molecule reacts with the very same surface. This means, the analyte and its copies should not stick to the surface to enable the efficient enzyme catalyzed polymerization, but “bends” towards this surface in order to react with the second probe. This structure sparked the name “bridge amplification”. The bridges spread across the same surface. Initial seed islands grow geometrically which means that the further extension occurs predominantly along their edges and the effective amplification efficiency decreases progressively [Mercier, 2003; Adessi, 2000].
Beside optical detection of molecular recognition events which are predominantly based on corresponding labeling methods alternative approaches exist which are exploiting the electrical properties of biomolecules. Capacitive biosensors were disclosed in U.S. Pat. No. 5,532,128, US 20040110277 or WO 2009003208. Herein, the general principle uses changes in the dielectric properties which lead to a change of the capacitance of sensor elements. Measurements between closely spaced electrodes or conductors which form tiny nanometer sized gaps promise sensitivities high enough to detect very few molecules and even single molecules. The production of said gaps is technologically difficult but feasible. Techniques like electron beam lithography [Hwang, 2002], electrodeposition and -migration [Iqbal, 2005], composite layer build-up and etching [WO 2009003208] and different fracture techniques [Reed, 1997; Reichert, 2002] have been applied to separate two conductors by such nanometer sized gaps.
For the detection of molecules which have more than one recognition site it is advantageous to modify each of two opposite conductors with a different molecular probe. But, such kind of individual conductor modification is challenging when it comes to nanometer dimensions. In WO 2009003208 Steinmuller-Nethl et al. have proposed to use different materials for each conductor while the conductors are separated trough an insulating layer with a thickness of only several nanometers. The different conductor materials enable the successive and selective binding of the molecular probes. As the number of electrically conducting but different materials in line with a specific and effective binding chemistry is limited, such an approach is not applicable for building large arrays.
Gao and Chen [WO 2010104479 A1] made electrode-insulator-electrode sandwich assemblies with stepped arrangements of electrodes which are separated by few nanometers thick layer of for example silicon oxide. Here, the electrode array structures were made on one single carrier substrate before functionalizing them with the respective capture probes. The problem of selectively immobilizing said capture probes on one of two corresponding electrodes which are separated by just a tiny step in the order of few nanometers, experimentally realized were separation layers between 5 and 20 nm, has been likewise recognized to be impossible by means of robotic spotters. The chosen method involves the binding of thiol-functionalized probes to all gold-electrodes, the selective removal by electrochemical stripping and repeated binding of thiol-functionalized probes to the second gold-electrode, and so forth. Each functionalizing requires 2 hours for the binding step alone plus the time which is needed for additional stripping and washing steps. Such method is unsuitable to build complex sensor arrays because it is very time consuming and prone to cross-contamination when once functionalized capture probes must be stripped to obtain pristine surfaces again with all previously bound molecules being physically removed before introducing the new species. This approach suffers from low scalability. Lee and Moon [EP 2088430 A1] presented assemblies of conducting elements like metallic nanowires which were crafted to either one upper or one lower substrate surface. Both such electrode have significantly enlarged nanoscopic surface areas. Here, the upper and the lower electrode carrying the same capture probes, e.g. one antibody. It is intended that the corresponding inserted antibody binds to each of the surfaces separately. It is intended to neither modify the surfaces separately nor to design complex sensor arrays by this method.
Thus, the double sided functionalizing of nanogap capacitor arrays with different biomolecules remains an unsolved issue.
From the state of the art the publications WO 2010/1204479 and EP 2088430 are known. WO 2010/1204479 is direct to a sensor for detecting a nucleic acid molecule comprising an electrode arrangement with two electrodes and nucleic acid probes immobilized at the surface of the electrodes. The present invention also refers to a kit and a method of using the sensor or a sensor array. The present invention is further directed to a process of manufacturing a sensor and sensor array.
EP 2088430 provides a bio-sensor including nanochannel-integrated 3-dimensional metallic nanowire gap electrodes, a manufacturing method thereof, and a bio-disk system comprising the biosensor. The biosensor includes an upper substrate block having a plurality of metallic nanowires formed on a lower surface thereof and including an injection port through which a biomaterial containing sample is injected, a lower substrate block having a plurality of metallic nanowires formed on an upper surface thereof, and a supporting unit supporting the upper and lower substrate blocks so that the upper and lower substrate blocks can be disposed spaced apart at a predetermined distance to form a nanochannel, wherein the metallic nanowires formed on the upper and lower substrate blocks are combined to form three-dimensional metallic nanowire gap electrodes.